The Role of Acanthamoeba culbertsoni Serine Proteases in Abating Microglial-Like Cell Cytokines ...

98 Pages·2016·1.67 MB·English

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Theses and Dissertations Graduate School
2009
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Jenica Harrison
Virginia Commonwealth University
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THE ROLE OF ACANTHAMOEBA CULBERTSONI SERINE PROTEASES IN
ABATING MICROGLIAL-LIKE CELL CYTOKINES AND CHEMOKINES

A dissertation submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy at Virginia Commonwealth University.

by

JENICA LEDAH HARRISON
B.S., Western Carolina University, 2000

Director: GUY A. CABRAL, PHD
PROFESSOR, DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY

Virginia Commonwealth University
Richmond, Virginia
April, 2009
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Acknowledgements

I would like to thank the following individuals for their support during my
graduate career. Without your help I would not have been able to complete this program.
A special thanks to my Lord and Savior Jesus Christ who has walked with me
every step of my journey.
I am grateful to my advisor, Dr. Guy A. Cabral for allowing me to pursue my
research in his laboratory. Over the years, he has taught me the importance of “allowing
the data to speak” to the direction of the research. Most importantly, he has also taught
me how to be an upstanding research scientist. I would also like to thank Dr. Francine
Marciano-Cabral for all of her guidance and assistance with my research project.
I would like to thank my other graduate committee members; Dr. Kathleen
McCoy, Dr. Michael McVoy, and Dr. Sandra Welch for their advice and interest in my
research. I would also like to thank Dr. Joy Ware and Dr. Dan Huang for teaching me the
two-dimensional (iso-dalt) gel electrophoresis technique.
I am thankful to the National Institutes of Health for their financial support while
pursuing my graduate degree.
I would also like to thank Daniel Southern and Dr. Christine Stevens of the
Clinical Laboratory Sciences Program at Western Carolina University for introducing me
to clinical Microbiology and Immunology and encouraging my interest in research.
While pursuing this graduate degree I have had the honor of working with several
people in the Cabral and Marciano-Cabral laboratories that I would like to thank: Dr.
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Tammy Ferguson, Dr. Angela Fritzinger, Dr. Andrea Staab, Dr. Rebecca MacLean, Dr.
Erinn Raborn, Dr. Gabriela de Almeida Ferreira, Dr. Bruno da Rocha Azevedo, Dr.
Daniel Fraga, Dr. LaToya Griffin-Thomas, Christina Hartman, Melissa Jamerson, Alex
Mensah, and Olga Tavares-Sanchez.
Last, but definitely not least, I could like to thank my family; my mother Jeanette
Harrison, my father Jerry Harrison (deceased), my grandmother Viola M. Harrison
(deceased), Joyce Jefferson, Jasmin Bhalodia, Ronald Saunders, William Day, and
Deborah Day. Thanks for your prayers, advice, guidance, and love. You all have been
an integral part of my life and for that I am forever grateful.
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Table of Contents

Page
Acknowledgements ............................................................................................................. ii
List of Tables ..................................................................................................................... vi
List of Figures .................................................................................................................. vii
Abstract ...............................................................................................................................x
Introduction..........................................................................................................................1
Materials and Methods .......................................................................................................17
Reagents ......................................................................................................17
Amoebae ......................................................................................................17
Microglia .....................................................................................................18
Conditioned Medium ...................................................................................18
Two-Dimensional (Iso-Dalt) Gel Electrophoresis (2D-PAGE) ..................20
Gel Zymography ..........................................................................................21
Light Microscopy ........................................................................................22
Multiprobe Ribonuclease Protection Assay (RPA) .....................................22
Cytokine/Chemokine Protein Microarray ...................................................23
Enzyme-Linked Immunosorbent Assay (ELISA) .......................................24
Data Analysis ..............................................................................................25
Results...............................................................................................................................26
v

Discussion…………………………………………………………………....................57
Literature Cited……………………………………………………………....................69
vi

List of Tables
Page
Table 1: Genotypes of Acanthamoeba Associated with Disease in Humans...................4
vii

List of Figures

Page
Figure 1: Life Cycle Forms of Acanthamoeba ....................................................................2
Figure 2: Characteristic Granuloma Associated with Granulomatous Amoebic
Encephalitis (GAE) ..............................................................................................8
Figure 3: Visualization of Complete Neurobasal™-A Medium Proteins ..........................27
Figure 4: Visualization of A. culbertsoni-Conditioned Medium Proteins .........................28
Figure 5: A Comparison of the Effect of Propagation Medium on A. culbertsoni
Protease Activity ................................................................................................30
Figure 6: Titration of Protease Activity in 109 A. culbertsoni-Conditioned Medium ........31
Figure 7: Acanthamoeba culbertsoni Secrete Proteases that Are Inhibited by the
Serine Protease Inhibitor Phenylmethylsulphonylfluoride (PMSF) ...................33
Figure 8: Acanthamoeba culbertsoni Secrete Proteases that Are Not Inhibited by the
Cysteine Protease Inhibitor trans-Epoxysuccinyl-L-leucylamido
(4-guanidino)butane (E-64) ................................................................................34
Figure 9: A Comparison of the Proteolytic Activity in Three Different Types
of A. culbertsoni-Conditioned Media .................................................................35
Figure 10: Acanthamoeba culbertsoni-Conditioned Medium Proteolytic Activity
Increases in a Time-Related Manner and Is Augmented During Co-Culture
With BV-2 Cells ...............................................................................................37
viii

Figure 11: Acanthamoeba culbertsoni Induces Chemokine mRNA Expression by
BV-2 Cells ........................................................................................................38
Figure 12: Cytokine and Chemokine Protein Expression by BV-2 Cells following
9 h Co-culture with A. culbertsoni. ...................................................................40
Figure 13: Cytokine and Chemokine Protein Expression by BV-2 Cells following
24 h Co-culture with A. culbertsoni ..................................................................41
Figure 14: Light Microscopy Images of BV-2 cells, A. culbertsoni, and BV-2 cells
Plus A. culbertsoni Co-cultures ........................................................................43
Figure 15: Conditioned Medium from A. culbertsoni Degrades Constitutively
Expressed Cytokines and Chemokines Elicited by BV-2 Cells........................45
Figure 16: Effect of 109 A. culbertsoni-Conditioned Medium (ACM-HI) on BV-2
Cell Morphology ...............................................................................................47
Figure 17: Conditioned Medium from A. culbertsoni Degrades Cytokines and
Chemokines Elicited from Lipopolysaccharide (LPS) Stimulated BV-2
Cells ..................................................................................................................48
Figure 18: Acanthamoeba culbertsoni Proteases Degrade A. culbertsoni Induced
Microglial Chemokines from BV-2 cells ..........................................................50
Figure 19: Acanthamoeba culbertsoni Protease Activity Persists in Co-culture
Supernatants Following Incubation for 72 h .....................................................52
Figure 20: Acanthamoeba culbertsoni Serine Proteases Degrade TNF-α .........................53
Figure 21: Acanthamoeba culbertsoni Serine Proteases Degrade MIP-1α ........................54